AbstractThe cytosolic enzyme uroporphyrinogen decarboxylase (URO‐D) catalyzes the fifth step in the heme biosynthetic pathway, converting uroporphyrinogen to coproporphyrinogen by decarboxylating the four acetate side chains of the substrate. Recombinant human URO‐D has been expressed in Escherichia coli with a histidine tag and has been purified to homogeneity. Purified protein was determined to be a monodisperse dimer by dynamic light scattering. Equilibrium sedimentation analysis confirmed that the protein is dimeric, with a dissociation constant of 0.1 μM. URO‐D containing an amino‐terminal histidine tag was crystallized in space group P3121 or its enantiomer P3221 with unit cell dimensions a = b = 103.6 Å, c = 75.2 Å. There is one molecule in the asymmetric unit, consistent with generation of the dimer by the twofold axis of this crystallographic operator. Native data have been collected to 3.0 Å resolution.